Group B Streptococcus (GBS) resides in the genitourinary tract and colonizes up to 35% of pregnant women contributing to chorioamnionitis, preterm birth, stillbirth, and neonatal sepsis and meningitis. GBS infections occur in utero following invasion of the placental membranes or during childbirth upon aspiration within the vaginal canal. Our prior studies have shown that specific GBS genotypes are more common among neonatal disease cases than pregnant mothers with a superior ability to infect cells at the maternal-fetal interface. However, there are critical gaps in our understanding of the mechanisms that different GBS strains use to persistently colonize and/or invade the placental membranes. We expect that phenotypic variation and its impact on host responses is an important yet understudied contributor to consider and represents the focus of this proposal. Our preliminary data show that distinct GBS strains secrete extracellular membrane vesicles (MVs) that vary in quantity, size and protein content and that these MVs elicit inflammatory cytokine responses from macrophages in vitro. We also found that MVs from two different GBS strains contained differentially abundant proteins that were classified as known and novel GBS virulence factors, suggesting that MVs play a role in GBS pathogenesis. Although MVs were recently described in GBS, only one control strain of a rare genotype was studied. Consequently, we propose to: 1) Determine the influence of GBS MVs on host response in human gestational tissues; 2) Determine the influence of MVs on host response and fetal outcome in pregnant mice; and 3) Ascertain the protein content of GBS MVs. We will characterize the content of MVs from both invasive and non-invasive strains of varying genotypes and classify host responses from primary cells recovered from the maternal-fetal interface as well as following infection of pregnant mice. We predict that MVs from the invasive GBS strains and not the commensal strains will trigger inflammatory responses that contribute to fetal or neonatal injury. In the short term, we will determine the role that MVs play in inflammatory responses, adverse pregnancy outcomes and virulence. Our long-term goal is to discover novel biomarkers associated with infection, vaccine targets and delivery systems that can potentially impact diseases caused by GBS and other pathogens with similar lifestyles.